The Pr55Gag protein of the human immunodeficiency virus type I (HIV-1) contains all of the information required for transport to assembly sites on the plasma membrane, association with genomic RNA, and release into extracellular space (see, e.g., Swanstrom, R. & Wills, J. W., 1997, in Retroviruses, eds. Coffin, J. M., Hughes, S. H. & Varmus, H. E., Cold Spring Harbor Laboratory Press, New York, pp. 263-334). However, although Gag is sufficient for viral assembly, cellular proteins are likely to facilitate the process. Several cellular proteins can be recovered from purified virions, suggesting proximity to the assembling particle (see, e.g., Arthur, L. O., Bess, J. W., Jr., Sowder, R. C. I., Benveniste, R. E., Mann, D. L., Chermann, J.-C. & Henderson, L. E., 1992, Science 258, 1935-1938; and Ott, D. E., Coren, L. V., Kane, B. P., Busch, L. K., Johnson, D. J. Sowder, R. C. I., Chertova, E. N., Arthur, L. O. & Henderson, L. E., 1996, J Virol. 70, 7734-7743). Others interact directly with Pr55Gas (see, e.g., Luban, B., Bossolt, K. L., Franke, E. K., Kalpana, G. V. & Goff, S. P., 1993, Cell 73, 1067-1078). Interaction with still others is implied, since Gag contains post-translational modifications (see, e.g., Bryant, M. & Ratner, L., 1990, Proc. Natl. Acad. Sci. USA 87, 523-527; Camaur, D., Gallay, P., Swingler, S. & Trono, D., 1997, J. Virol. 71,6834-6841; Göttlinger, H. G., Sodroski, J. G. & Haseltine, W. A., 1989, Proc. Natl. Acad. Sci. USA 86,5781-5785; Ott, D. E., Coren, L. V., Copeland, T. D., Kane, B. P., Johnson, D. G., Sowder, R. C., 2nd, Yoshinaka, Y., Oroszlan, S., Arthur, L. O. & Henderson, L. E., 1998, J. Virol. 72, 2962-2968). There are now reports that the region in Gag required for release of mature particles, the late (L) domain (see, e.g., Wills, J. W., Cameron, C. E., Wilson, C. B., Xiang, Y., Bennett, R. P. & Leis, J., 1994, J. Virol. 68, 6605-6618; Göttlinger, H. G., Dorfman, T., Sodroski, J. G. & Haseltine, W. A., 1991, Proc. Natl. Acad Sci. USA 88, 3195-3199; Huang, M., Orenstein, J. M., Martin, M. A. & Freed, E. O., 1995, J. Virol. 69, 6810-6818) directs the interaction of the protein with the ubiquitination machinery (see, e.g., Schubert, U., Ott, D. E., Chertova, E. N., Welker, R., Tessmer, U., Princiotta, M. F., Bennink, J. R., Krausslich, H. G. & Yewdell, J. W., 2000, Proc. Natl. Acad. Sci. USA 97, 13057-13062; Strack, B., Calistri, A., Accola, M. A., Palu, G. & Gottlinger, H. G., 2000, Proc. Natl. Acad. Sci. USA 97, 13063-13068; Vogt, V. M., 2000, Proc. Natl. Acad Sci. USA 97, 12945-12947; Ott, D. E., Coren, L. V., Chertova, E. N., Gagliardi, T. D. & Schubert, U., 2000, Virology 278, 111-121, Patnaik, A., Chau, V. & Wills, 3. W., 2000, Proc. Natl. Acad Sci. USA 97, 13069-13074). Based on its sequence and recent studies, Tsg101 is an ubiquitin (Ub)-conjugating E2 enzyme variant (UEV) protein involved in regulation of intracellular trafficking, transcriptional regulation, and cell cycle control (see, e.g., Babst, M., Odorizzi, G., Estepa, E. J. & Emr, S. D., 2000, Traffic 1,242-258; Lemmon, S. K. & Traub, L. M., 2000, Curr. Opin Cell Biol. 12,457-466, Xie, W., Li, L. & Cohen, S. N., 1998, Proc. Natl. Acad. Sci. USA 95, 1595-1600; Zhong, Q., Chen, Y., Jones, D. & Lee, W. H., 1998, Cancer Res. 58,2699-2702; Sun, Z., Pan, 3., Hope, W. X., Cohen, S. N. & Balk, S. P., 1999, Cancer 86,689- 96). UEV proteins lack the critical Cys residue essential for conjugation and transfer of Ub to protein substrates or Ub-ligating (E3) enzymes (Koonin, E. V. & Abagyan, R. A., 1997, Nat. Genet. 16,330-331; Ponting, C. P., Cai, Y.-D. & Bork, P., 1997, J. Mol. Med. 75,467-469). They are highly conserved in evolution and constitute a novel family of proteins structurally related to, but distinct from, E2 enzymes.
All retroviruses have in common 3 genes, gag, pol, and env, which specify the structural and enzymatic functions of the virus (see, e.g., Swanstrom, R. & Wills, J. W., 1997, in Retroviruses, eds. Coffin, J. M., Hughes, S. H. & Varmus, H. E., Cold Spring Harbor Laboratory Press, New York, pp. 263-334). The gag gene alone is sufficient for assembly and release of immature virus-like particles from infected cells. Maturation to form the infectious particle requires a viral-encoded protease (PR) encoded in pol. The gag-encoded protein (Gag) contains distinct domains involved in assembly and release. There is a plasma membrane-binding (M) domain located in the N-terminal matrix (MA) region, a capsid (CA) domain that forms a genome-encasing core structure, a protein interaction (I) domain in the nucleocapsid (NC) region, and a late (L) domain, required for release by budding from the plasma membrane. The L domain is a Pro-rich motif that is highly conserved in retroviruses; other enveloped viruses, including rhabdo-, fib-, and Epstein Barr viruses, and cellular proteins also have Pro-rich motifs (Harty, R. N., Brown, M. E., Wang, G., Huibregtse, I. & Hayes, F. P., 2000, Proc. Natl. Acad Sd. USA 97, 13871-13876; Wills, J. W., Cameron, C. E., Wilson, C. B., Xiang, Y., Bennett, R. P. & Leis, J., 1994, J Virol. 68, 6605-6618; Göttlinger, H. G., Dorfman, T., Sodroski, J. G. & Haseltine, W. A., 1991, Proc. Natl. Acad Sci. USA 88, 3195-3199; Huang, M., Orenstein, J. M., Martin, M. A. & Freed, E. O., 1995, J. Virol. 69, 6810-6818; Ikeda, M., Ikeda, A., Longan, L. C. & Longnecker, R., 2000, Virol. 268, 178-191; Yasuda, J. & Hunter, E., 1998, J. Virol. 72, 4095-4103; Harty, R. N., Paragas, J., Sudol, M. & Palese, P., 1999, J. Virol. 73, 2921-2929). The L domains in retroviruses differ in amino acid sequence and location within the respective viral structural proteins, but are functionally exchangeable (Wills, J. W., Cameron, C. E., Wilson, C. B., Xiang, Y., Bennett, R. P. & Leis, J., 1994, J. Virol. 68, 6605-6618; Parent, L. J., Bennett, R P., Craven, R. C., Nelle, T. D., Krishna, N. K., Bowzard, J. B., Wilson, C. B., Puffer, B. A., Montelaro, R. C. & Wills, J. W., 1995, J. Virol. 69, 5455-5460), suggesting commonality of function.
The protein product of the TSG101 gene was originally identified by the reversible neoplasia associated with its functional inactivation in murine fibroblasts (Li, L. & Cohen, S. N., 1996, Cell 85, 319-329). Sequence analysis has suggested (Li, L. & Cohen, S. N., 1996, Cell 85, 319-329; Koonin, E. V. & Abagyan, R. A., 1997, Nat. Genet. 16,330-331; Ponting, C. P., Cai, Y.-D. & Bork, P., 1997, J. Mol. Med. 75, 467-469), and experimental evidence has shown, that Tsg101 can function in both the modulation of transcription (Sun, Z., Pan, J., Hope, W. X., Cohen, S. N. & Balk, S. P., 1999, Cancer 86, 689-96; Watanabe, M., Yanagi, Y., Masahiro, Y., Yano, T., Yoshikawa, H., Yanagisawa, J., & Kato, S., 1998, Biochem, Biophys. Res. Commun. 245, 900-905; Hittleman, A. B., Burakov, D., Iniguez-Lluhi, J. A., Freedman, L. P., & Garabedian, M. J., 1999, EMBO 3, 18, 5380-5388) and the inhibition of ubiquitination and protein decay (Li, L., Liao, J., Ruland, J., Mak, T. W., & Cohen, C. N., 2001, Proc. Natl. Acad. Sci. USA 98, 1619-1624). The latter effects are mediated by an N-terminal region that contains a ubiquitin (Ub) conjugase (E2)-like domain, but lacks an active site Cys residue crucial to Ub conjugation. The structure of the UEV domain of Tsg101 and its modes of interaction with Ub and PTAP (SEQ ID NO: 39) have also been reported (Pornillos et al., 2002, EMBO J. 21:2397-2406).
Cells deficient in Tsg101 show a variety of nuclear, microtubule, and mitotic spindle abnormalities (Xie, W., Li, L. & Cohen, S. N., 1998, Proc. Natl. Acad. Sci. USA 95, 1595-1600; Zhong, Q., Chen, Y., Jones, D. & Lee, W. H., 1998, Cancer Res. 58, 2699-2702), and tsg101 null mutant mice show defective cell proliferation and early embryonic death (Ruland, J., Sirard, C., Elia, A., MacPherson, D., Wakeham, A., Li. L., de la Pompa, J. L., Cohen, S. N., & Mak, T. W., 2001, Proc. Natl. Acad. Sci. USA 98, 1859-1864). The steady state level of Tsg101 normally is controlled post-translationally within a narrow range in cultured murine and human cell lines (Feng, G. H., Lih, C.-J., & Cohen, S. N., 2000, Cancer Research 60, 1736-1741) and over-expression from an adventitious promoter, as well as tsg101 deficiency, can lead to neoplastic transformation (Li, L. & Cohen, S. N., 1996, Cell 85, 319-329). Regulation of the Tsg101 protein level, which can be affected by the Ub ligase, Mdm2 (Li, L., Liao, J., Ruland, J., Mak, T. W., & Cohen, S. N., 2001, Proc. Natl. Acad. Sci USA 98, 1619-1624), requires a C-terminal Tsg101 sequence that is evolutionarily highly conserved in organisms as disparate as humans, C. elegans, S. pombe, and D. melanogaster (Feng, G. H., Lih, C.-J., & Cohen, S. N., 2000, Cancer Research 60, 1736-1741; Bishop N and Woodman P., 2001, J Biol Chem. 276:11735-42).
Stp22p/Vps23, a class E vacuolar protein sorting (vps) protein in S. cerevisiae, has been identified as a Tsg101 orthologue and Tsg 101 itself has been implicated in the trafficking of membrane-associated proteins (Bishop N and Woodman P., 2001, J Biol Chem. 276:11735-42; Babst, M., Odorizzi, G., Estepa, E. J. & Emr, S. D., 2000, Traffic 1, 242-258; Li, Y., Kane, T., Tipper, C., Spatrick, P. & Jenness, D. D., 1999, Mol. & Cell. Biol. 19, 3588-3599). It has also been shown that SL6 cells are defective in sorting of multiple surface-bound proteins (Babst, M., Odorizzi, G., Estepa, E. J. & Emr, S. D., 2000, Traffic 1, 242-258).
Citation of references hereinabove shall not be construed as an admission that such references are prior art to the present invention.